Super-resolution microscopy (PALM)

A key technique for our team is the well-known PhotoActivated Localization Microscopy (PALM). With this imaging method, invented in 2006 [1-3] we can use and characterize the phototransformable fluorescent proteins we develop for advanced fluorescence imaging applications. The principle of PALM is to circumvent the diffraction-limited optical resolution ( 200-300 nm) thanks to the observation of densely labeled samples, a few molecules at a time. The photoactivation of a very limited number of individual molecules per acquired frame (typically with a time exposure of a few ms), allow their precise localization because of their spatial separation. The accumulation of thousands of such frames, each containing a small number of single molecules that are observed until photobleached will permit the reconstruction of a subdiffraction image ( 20 nm resolution), also called super-resolution image.
Our home-made setup is equipped with five CW solid-state lasers whose wavelengths are ranging from violet to red and intensities between 50-400 mW. The output of these lasers is regulated by a computer-controlled acousto-optical tunable filter (AOTF) and a white lamp is also useable for specific wavelengths. Our motorized inverted microscope (Olympus IX81) is equipped with several objectives with magnification ranging from 5X to 100X, which can be coupled to an anti-drift “nose-piece” system (Olympus). The illumination of the sample is quickly switchable between direct wide-field and total internal reflection fluorescence (TIRF) modes. A fiber-coupling to a spectrophotometer is also possible for a better spectral characterization of a sample along with the image acquisition. A special home-made detection system allows recording images with two EMCCD cameras (Photometrics Evolve 512) on one to four color channels simultaneously.(A) Example of PALM on a human cell. Actin fibers fused to a green-to-red photoconvertible fluorescent protein are observed in conventional wide field microscopy (left) and in PALM (right). The inset demonstrates the optical resolution improvement. (B) The PALM microscope room and a zoom on the optical table with our setup.